Functionally active platelets are of value both with regard to diagnosis and with regard to therapy. An example of an aspect of using a preparation of functionally active platelets is that of employing the preparation as control material for platelet function diagnosis. Aggregometry plays the most important part in this diagnosis. The diagnosis involves measuring the reaction of fresh platelets, which are as a rule present as platelet-rich plasma, to various inducers, in particular ADP, adrenaline, arachidonic acid, collagen and thrombin. The reaction is normally measured using the known turbidimetric methods. The inducers (apart from adrenaline) first of all bring about a change in the shape of the cells, with this change being recognizable by a transient increase in the extinction. Aggregation subsequently takes place. Biphasic aggregation curves often result. The second phase is closely linked to the release reaction and to prostaglandin synthesis. Arachidonic acid and collagen induce only monophasic aggregation curves as an expression of irreversible aggregation.
The investigation of platelet function has nowadays been extensively automated for reasons of rationalization and precision. It is absolutely necessary to have a quality control strategy for assessing the results. Hitherto, the only option for controlling this diagnosis has been that of using a pool of samples from "normal" donors (Abernathy et al. (1978) Throm.Haemostas. 39, p. 246).
For a laboratory, this is a control material which can only be prepared with a great deal of effort and expense. While a stable and functionally active platelet preparation having defined and constant properties is desirable for controlling platelet function, such a preparation has not hitherto been available.
Another aspect of the use of stable, functionally active platelets is their use in diagnostic test methods in which platelets are one of the reagents to be employed in the method. One example of such methods are tests for diagnosing heparin-associated thrombocytopenia (HAT). This is a rare (approx. 5%) but serious complication of antithrombotic therapy with heparins. Nonimmunological forms (HAT I) are distinguished from immunologically determined forms (HAT II). In contrast to other medicinally induced thrombocytopenias, which as a rule provoke bleeding complications, HAT results in thromboembolic complications which can extend to the occlusion of large blood vessels. Various diagnostic approaches are used for the early diagnosis of a type II HAT. Apart from counting platelets, which is an initial exploratory test, and the serotonin release test as the reference method, a test for heparin-induced platelet aggregation (HIPA) is particularly suitable, which test approaches the reference method in sensitivity and specificity (Greinacher et al. (1991) Thromb.Haemostas.6616),734(1991) Greinacher et al. (1994) Transfusion 34, p. 381). This test investigates whether the patient plasma can cause thrombocytes from healthy donors to aggregate under suitable conditions (low heparin concentration). The preparation of the thrombocytes, which necessitates the pooling of platelet-rich plasma from several healthy donors, is very expensive and time-consuming for a laboratory and has previously stood in the way of introducing the test into laboratory routine. A preparation of stable thrombocytes which are still functionally active to the extent that they can be aggregated under suitable conditions would substantially simplify and accelerate implementation of this test.
Therapeutically, platelet concentrates can be used for treating disturbances of platelet function which are of varying origin. Platelet concentrations of&lt;70,000 per microliter are referred to as thrombocytopenias. Thrombocytopenias are due either to insufficient production of platelets, to an accelerated degradation of these cells or to abnormal distribution (Colman et al. (1987) Haemostasis and Thrombosis (J. B. Lipincott Co.) 2nd Ed., Chapter 28). In principle, inadequate platelet function can be genetically determined (e.g.: Glanzmann's thrombasthenia or Bernard-Soulier syndrome) or be acquired (e.g. failure of the bone marrow in association with malignant diseases, chemotherapy or disseminated intravasal coagulation). A number of drugs, pharmaceuticals and ionizing radiations can lead to acquired thrombocytopenias.
Patients who suffer from a thrombocytopenia have a bleeding tendency which is similar to hemophilia. As a rule, the bleeding is from capillary blood vessels; a typical example is relatively minor bleeding into the mucous membranes (petechiae). Normally, minor damage to the capillary vessel wall is sealed by agglutinating platelets.
Nowadays, patients having low platelet numbers are treated by infusion of platelet concentrates. These concentrates typically contain 6.times.10.sup.10 platelets in approx. 50 ml of plasma. They are prepared by centrifuging anticoagulated blood in a stepwise manner and taking up the platelet sediment once again into plasma. Alternatively, platelet concentrates can be prepared using an apheresis apparatus, which separates the platelets directly from blood. Under suitable conditions (room temperature), platelet concentrates will keep for up to 7 days. During storage, the bags which contain the concentrate have to be maintained constantly in motion.
Although it would be desirable if the platelet concentrates kept for a longer period, it has not previously been possible to achieve this.
A first possible strategy for increasing the durability of platelet concentrates for diagnostic or therapeutic purposes consists in switching off particular mechanisms for activating the platelets. This is intended to ensure that the platelets are not prematurely activated, by the process of their being enriched and stored, to secrete ingredients and to aggregate. Various strategies of this nature are described in the literature; these strategies extend from defined washing procedures through to the addition of specific inhibitors:
Calcium, which is an activator of platelet aggregation, can be complexed by a chelating agent, for example EDTA. PA1 The activator ADP can be broken down completely to AMP by adding the enzyme apyrase. PA1 Plasma factors which might contribute to platelet activation can be removed by washing the platelets. PA1 Thrombin can be inhibited by adding hirudin or heparin/antithrombin III. PA1 The addition of prostacyclin (PGI.sub.2) prevents platelet aggregation by means of stimulating the cellular adenylate cyclase. PA1 Aspirin or indomethacin inhibit the cyclooxygenase of the platelets and thereby irreversibly switch off the route for synthesizing thromboxane. PA1 Glucose at a concentration of between 0 g/l and 100 g/l, preferably of from 1 g/l to 10 g/l, particularly preferably of about 2.4 g/l, PA1 a magnesium salt, preferably magnesium chloride, at a concentration of between 0 g/l and 100 g/l, preferably of from 1 g/l to 5 g/l, particularly preferably of about 1.2 g/l, PA1 a potassium salt, preferably potassium chloride, at a concentration of between 0 g/l and 100 g/l, preferably of from 1 g/l to 5 g/l, particularly preferably of about 1.6 g/l, PA1 a sodium salt, preferably sodium chloride, at a concentration of between 0 g/l and 100 g/l, preferably of from 0.5 g/l to 5 g/l, particularly preferably of about 0.6 g/l.
However, it has been found in practice that adding the inhibitors irreversibly damages the platelets or inhibits them powerfully in their function. There is a need for a method for stabilizing these cells in a gentle manner without significantly impairing their function.
Any stabilizing method must ensure that the platelets retain a certain degree of functional activity throughout the entire process; this includes ensuring, for example, that the platelets do not alter their shape, do not excrete activators and do not aggregate during the preparation and stabilization of the concentrate. In a stable platelet preparation, the platelets should be present as individual cells having a predominantly discoid shape. The functional activity presupposes the retention of certain cell organelles (e.g. a-granula) and, at the molecular level, the retention of certain receptors on the cell-surface, for example glycoprotein Ib/IX, which serves as the receptor for the von Wille-brand factor, or glycoprotein IIb/lIIa, which serves as the receptor for fibrinogen. It is furthermore necessary for certain metabolic pathways which release messenger substances in response to the binding of ligands to the receptors and which set in motion physiological processes, for example secretion from a-granula, to remain intact.
Platelets which have been stabilized by freeze-drying have already been described, although these platelets only react in an appropriate manner to activation by von Willebrand factor.
Consequently, the present invention was based on the object of making available a platelet preparation which, meets the above-described requirements.